9. Mixed And Pure Cultures

Question 1

what is a pure culture?

Answer 1

• labratory culture containing a single species of microorganism

Question 2

what is a mixed culture?

Answer 2

• culture contianing 2 or more species of microorganisms

Question 3

what is the need to isolate a pure culture?

Answer 3

• mass quatity needed for detailed studies
• generate strains of interest
• produce specific products

Question 4

what methods are used to obtain a pure culture?

Answer 4

• streaking
• serial dilution

Question 5

what is the process of streaking?

Answer 5

• mixed culture of bacteria streaked along a nutrient medium
• does use different streaking patterns

Question 6

what is the process of serial dilution?

Answer 6

• pour-plate technique of isolation
  – inoculation of empty plate
  – addition of melted agar nutrient
  – swirl to mix
  – colonies grow in and on solidified medium
• spread plate method
  – inoculate plate containing solid medium
  – spread inoculum over surface evenly
  – colonies grow only on surface of medium

Question 7

what are the aims of keeping a culture pure?

Answer 7

• prevent / reduce contamination from working environment

Question 8

how do you keep your sample pure?

Answer 8

• aseptic technique
  – flame sterilisation
  – reduction time of contact with air
  – autoclave ,air filter sterilisation, and etOH for equipment sterilisation
  – ensuring clean working environment

Question 9

what are the uses of pure cultures in biotech and bioprocesses?

Answer 9

• biochemical characterisation
• growth kinetics characterisation
• utilisation of single substrate; production of know metabolites/products
• monitoring processes for contamination

Question 10

what are the uses of mixed cultures in biotech and bioprocesses?

Answer 10

• robustness
  – more protection against contamination and sensitivity to constituents of influent stream
  – reduced infections for fermentations
• co-operative metabolism and metabolic trad-offs
• potentially enable use of cheap, impure substrates

Question 11

what are cultures mantained and stored for?

Answer 11

• retain viability
• avoid contamination
• eliminate genetic change

Question 12

how are cultures mantained?

Answer 12

• agar slant/plate storage
  – culture grown on slope mayve be in refrigerator (4 degrees)
  – sub-culture at approxiamately 6 month intervals

Question 13

what are the disadvantages to the traditional approach?

Answer 13

• risk of contamination
  – repeated rounds of sub-culturing; isolates contaminated by overgrown contaminants
• loss of viability
  – sub-culturing not at required intervals and inadequately stored; sensitive isolates irrecoverable
• genetic drift and mutation
  – sub-culture carries potential for genotypic and phenotypic changes; loss of virulence and resistance factors/reduced motility

Question 14

how are cultures stored at reduced temperatures?

Answer 14

• liquid nitrogen
• ultra freezer conditions (-80 degrees)
• freeze-drying

Question 15

how are cultures maintained through freeze drying?

Answer 15

• broth cultures place in freeze-drier
  – connected to vacuum pump
• vacuum removes all water from cells
  – forms powder
• standard practice for large commercial and national culture collections
  – not suitable for smaller labs; so no small stock culture collection